The most widely used strategy for determining the sequence of a fairly large segment of cloned DNA is to first generate a random overlapping population of fragments encompassing that piece and to then "shotgun" clone these fragments into a vector. The resulting pool of recombinants is called a "library." These recombinants can be individually propagated, and the DNA sequence of the individual cloned inserts may then be determined. The DNA sequence obtained from individual cloned inserts can be combined to generate the DNA sequence of the entire segment of DNA.
A common vector for generating such libraries is M13 phage, a filamentous bacteriophage that infects E. coli to produce progeny phage particles harboring only one strand (the "plus" strand) of the viral genome. The genomes of filamentous E. coli phage consist of single-stranded circular DNAs of about 6,400 nucleotides. The products of the genes encoded in this DNA are involved in phage DNA replication, phage capsid synthesis, and phage assembly.
The single strand produced by the M13 phage is an ideal template for oligonucleotide-directed DNA sequencing using the Sanger chain termination method (for example, Heidecker, et al., Gene, 10:68, 1980) because the method depends on the annealing of a oligonucleotide primer to a single-strand template. By using the M13 phage as a vector for a segment of cloned DNA, the cloned DNA can be produced in the single-strand form attached to known flanking sequences capable of being used as targets for the oligonucleotide primers.
After a fragment of DNA has been inserted into the filamentous phage, it is important to be able to differentiate between phage that have a cloned insert and phage that do not. The discrimination of clones containing inserted foreign fragments from those that do not is generally done by means of a blue-white color selection (J. Messing, in Methods of Enzymology 101 (part C), Wu et al., Eds, Academic Press, N.Y., 1983). The color selection technique works because the inserted DNA interrupts a gene responsible for producing an enzyme that produces a colormetric reaction when a specific substrate is added.
The blue/white selection technique is an "indirect" cloning approach because all phage (whether recombinant or not) are propagated. This approach, although powerful and convenient, is nonetheless laborious, costly, variable in efficiency, and refractory to effective automation.
Waye, et al. discloses a direct selection technique for the filamentous phage M13 using the properties of the bacterial EcoK gene.
What is needed in the art of cloning is a filamentous phage vector system that provides direct selection due to the properties of a filamentous phage gene, such as gene X. Gene X is a filamentous phage gene involved in phage DNA replication.